Pulmonary surfactant reduces surface tension at the air-liquid interface and has a crucial role in neonatal adaptation. With lung immaturity, a lack of surfactant leads to decreased pulmonary compliance and respiratory distress. The mainstay in management is oxygen and respiratory support which lead to oxidative injury and further pulmonary disease. Alveolar type II cells are critical for lung function because they secrete surfactant and repopulate the denuded epithelium after oxidative injury. Type Il cells also produce type IV collagen, an essential component of the extracellular matrix. Ascorbate (ASC) functions as an antioxidant and is critical for procollagen hydroxylation and secretion In preliminary results, ASC prevented oxidative injury in cultured rat type II cells as assessed by second messenger generation, surfactant secretion and cell viability. In peroxide-induced radical formation, ASC pretreatment significantly reduced spin adduct formation. This suggested ASC supported free radical reduction. In type II cells cultured from scorbutic guinea pigs, ASC was reduced from 4.5 nmol to 0.11 nmol per l0(-7) cells when compared to cells from control animals. Studies with this model suggested that cells with limited ASC availability were more susceptible to oxidative injury and that ASC pretreatment maintained viability. Protection due to nonspecific antioxidant properties of ASC were unlikely since analogues were not as effective in protecting the cell from oxidative injury. In this model, type I and IV collagen secretion and fibronectin secretion were significantly reduced, which suggested that ASC availability altered the basement membrane matrix. ASC addition to scorbutic cells restored secretion. In this proposal we will assess the functions of ASC in alveolar type II cells and its role in protection against oxidative injury. Using scorbutic type II cells as the model, we will determine if l) ASC supports free radical reduction, 2) limited ASC availability alters the basement membrane matrix and, subsequently, alters type II cell differentiation and 3) if secretion of surfactant apoproteins A and D, proteins that require hydroxylation for secretion, are altered by limited ASC availability. Since oxidative injury can decrease ASC, we will determine the extent to which ASC availability alters the repair process alter oxidative injury. The scorbutic cell provides a unique model to alter the extracellular matrix produced by the type II cell and to determine the subsequent affect on differentiation and repair alter oxidative injury. By assessing the ASC requirement in these processes, we can determine which processes are more severely affected by ASC deficiency and whether these are likely to be important at physiological concentrations in lung. These studies will provide important new knowledge about pulmonary ASC and its role in protection against oxidative injury.